Immunological disorders are manifested as a wide variety of diseases and pathologies, including autoimmune diseases, acute and chronic inflammatory disorders, organ transplant rejection, Graft-Versus-Host Disease (GVHD), lymphoid cell malignancies, septic and other forms of shock, loss of immune responsiveness as seen in HIV and SCIDS, and failure of the immune response to tumor growth.
Many immunological disorders are triggered by aberrant or uncontrolled responses to antigen. Autoimmune diseases are the result of the inappropriate response of the immune system to self-antigens, resulting in damage to cells and tissues. GVHD develops when donor cells from a bone marrow transplant (BMT) respond to host (i.e. patient) antigens. Organ transplant rejection results when the patient's immune system responds to antigens derived from the transplanted organ. Acute inflammatory disorders such as hyper-allergic conditions and shock are the result of uncontrolled immune response to the triggering antigens.
T cell dependent immune responses require T cell recognition of antigen. For example, GVHD results from a complex interplay between donor T cells and host immune system cells. The initiating event is the recognition by donor T cells of non-self, i.e. host, antigens. These are referred to as alloantigens. Alloantigen recognition by these donor cells results in the production of immunoregulatory and inflammatory cytokines and chemokines, which advance and exacerbate the donor anti-host immune response. This disease can develop in either an acute or chronic form, depending on the regulation of complex cytokine networks which control the type of immune response which develops. Immune responses can be characterized as Th0, Th1, or Th2 depending on 1) the nature of the cytokines and chemokines produced by activated T cells during the response, and 2) the nature of the cytokines and chemokines produced by accessory and other cells during the response. Examples of non-T cells important during immune responses are B cells, dendritic cells, monocytes and macrophages, follicular dendritic cells, and endothelial cells. Together the cytokines produced influence a variety of cell types to differentiate, and the chemokines produced influence cell trafficking and localization. Th0 responses are characteristic of the short term stimulation of previously unstimulated (naive) T cells. Th0 T cells produce moderate amounts of a number of cytokines, notably II-2 and TNF. Repetitively or chronically stimulated Th0 cells can differentiate into either Th1 or Th2 T cells, depending on a number of factors. Such factors include, but are not limited to, accessory cell cytokine production, the strength of T cell receptor engagement, and the nature of secondary signals received, e.g. via the CD28 costimulatory receptor. In particular exposure of activated T cells to the cytokine II-12, produced primarily by activated macrophages, supports differentiation to Th1 T cells, while exposure to II-4 and II-10 supports differentiation to Th2 T cells.
Th1 T cells produce cytokines such as II-2 and IFN-γ which are associated with inflammatory responses, T cell cytotoxicity, and macrophage activation. Th1 T cells respond to chemokines which attract cells into sites of tissue inflammation, such as Mip-1alpha, MIP-1beta, RANTES, IP-10, and MIG. Since cytotoxic T cells and activated macrophages act to eliminate damaged or infected cells, the Th1 response is responsible for controlling the immune response to intracellular pathogens. Importantly, production of Th1 chemoattractant chemokines such as IP-10 and MIG by macrophages and endothelial cells is closely regulated by IFN-y, which is the prototypic chemokine produced by Th1 T cells. Thus feedback loops may develop between activated T cells and their environment, which augment the development of a particular type of response at a particular time and location.
Th2 T cells produce cytokines such as II-4, II-5, II-6, and II-10 which support the development of humoral immune responses, including those which require the production of IgE, IgA, and IgG. These Ig responses are driven by the T cell mediated activation of B cells which “switch” their Ig phenotype from surface bound IgM and IgD to secreted Ig. Secreted Igs normally function to control infection from pathogens in circulation (IgG), at mucosal surfaces, such as the gut and oral cavity (IgA) and in the respiratory tract (IgE). Overproduction of Ig can cause disease, for example in SLE (IgG and IgA), allergic (Type I) hypersensitivity (IgE), and GVHD (IgG, IgA, and IgE). Th2 T cells also support the activation of eosinophils and Mast cells which can mediate acute responses to pathogens, for example in the respiratory tract. Th2 T cells respond to chemokines such as eotaxin and MDC, whose production is closely regulated by II-4, the prototypic cytokine produced by NK1.1 and activated T cells.
The interaction of T cells with B cells is a complex and closely regulated process. To begin the activation process, B cells must receive an antigen signal through the B cell antigen receptor (membrane Ig). Secondly, B cells must receive specific contact dependent and contact independent signals from activated T cells. One required contact dependent signal is delivered via the binding of CD40L on T cells to CD40 on B cells. One required contact-independent signal is delivered by II-4 secreted by activated T cells and by NK1.1 cells binding to the II-4 receptor on B cells. These signals appear to take place within the T cell areas of secondary lymphoid organs, such as the spleen. The spleen, lymph nodes, tonsils, Peyer's patches and other secondary and tertiary lymphoid organs have distinct microanatomical areas within which T and B cells typically reside. All lymphocytes migrate out of the blood or the lymph into the T cell area of these organs first, by crossing endothelial cell layers such as the High Endothelial Venules in lymph nodes and Peyer's patches and the marginal sinus endothelial cell layer in the spleen. Then, the B cells move into B cell areas known as B cell follicles, B cells which have traversed the T cell area but have not become activated will leave the follicle after a few days. Activated B cell undergo a process of differentiation. Some activated B cells, known as plasmacytes, secrete large amounts of antigen specific, low affinity IgM or IgG antibody. These B cells typically appear early after the induction of the immune response, and move into the red pulp of the spleen and other anatomical locations, where they persist, secreting antibody, for several days. Other activated B cells differentiate within a region of the follicle known as the secondary follicle, or germinal center. Germinal centers form around networks of specialized antigen retaining cells known as Follicular Dendritic Cells (FDC), which are thought to display antigen to drive or refine the germinal center reaction. B cells within the germinal center have “switched” their Ig phenotype, and undergo “affinity maturation”, with the result that they display a high affinity for their antigen target. Normally, the antigen target is a foreign antigen, although in diseases such as chronic GVHD and autoimmune disorders the Igs recognize self-antigens. Finally, B cells leave the follicles, migrate back through the T cells areas, and leave the organ via efferent circulation into the bloodstream.
B cells that have fully differentiated to express high affinity Ig are known as blast cells, and they leave B cell follicles to take up residence in various other environments, including the red-pulp areas of the spleen, the bone marrow, the liver, or mucosal cell layers lining the respiratory tract and gut. Some of these fully differentiated B cells are known as memory cells, and can persist for long periods of time, ready to respond to the same antigen if it is encountered again.
As B cells migrate from location to location within the lymphoid organs they require specific signals to guide them, and specific signals which ensure their survival. For example, multiple signals are required to maintain B cell follicular organization in the spleen. These include interaction of the BOA ligand with the chemokine receptor BLR-1, interaction of TNF with TNF-R55, and interaction of LTbeta with LTβ-R (reviewed in Chaplin and Fu, Current Opin. Immunol. 10: 298-297 (1998)). Mice which are deficient in any of these molecular pathways lose B cell follicular integrity in the spleen. Furthermore, all of these gene-deficient mice have also lost the ability to undergo germinal center reactions in the spleen. Disruption of other molecular pathways affects the germinal center reaction only. For example, mice deficient in CD40L maintain B cell follicles, but germinal centers do not form. B cells within the germinal center environment require signals through 0040 to maintain survival and to downregulate IgM and switch to Ig expression.
B cells move not only within the follicle and germinal center, but also leave the follicle after activation, and move to other sites within the body. Memory B cells can be found in the bone marrow, and B cells which express IgA specifically traffic to cell layers in the gut and other mucosal sites. Other signals presumably guide activated T cells to specific sites within and between lymphoid compartments. For example, cytotoxic T cell can migrate to the site of infection or other antigen challenge to find and lyse their targets. The identities of all the signals which guide T and B cells between different microanatomic locations are not yet known. However it appears that multiple pathways orchestrate the T and B cell responses to antigen, both in secondary lymphoid organs, and at the sites of infection or inflammation.
GVHD is a well studied example of an antigen driven immune response. GVHD is an often fatal consequence of bone marrow transplantation (BMT) in human patients. The disease can occur in an acute or in a chronic form. Acute and chronic forms of GVHD are prototypic examples of the development of antigen specific Th1 and Th2 responses, respectively. The acute form of the disease occurs within the first 2 months following BMT, and is characterized by donor cytotoxic T cell-mediated damage to skin, gut, liver, and other organs. The chronic form of the disease is manifested much later (over 100 days post-BMT) and is characterized by hyperproduction of immunoglobulin (Ig), including autoantibodies, and damage to the skin, kidney, and other organs caused by Ig-deposition. The development of acute GVHD is predictive of the subsequent development of chronic GVHD. Thus, the same patient can develop both diseases, in sequence. Approximately 50% of all BMT patients develop either acute or chronic GVHD. Nearly 90% of acute GVHD patients go on to develop chronic GVHD. No current therapies for chronic GVHD are successful in the majority of patients.
GVHD can be modeled in the mouse using parental into F1 cell transplantation regimens. In the model described here, splenocytes from the DBA2 strain of mice are injected iv into (DBA2×C57BI/6) F1 mice, which are referred to as B6D2F1. The injected splenocytes constitute the graft, and the DBA2 mouse is the donor of that graft. The F1 mouse which receives the graft is the host, Donor T cells present in the graft recognize half of the MHC markers (haplotypes) on host cells as foreign, because they are derived from the other, C57BI/6 parent, This induces a donor T cell response against the host resulting in GVHD. When DBN2 parental splenocytes are injected into the B6D2F1 host, chronic GVHD develops. In contrast when C57BI/6 splenocytes are injected into the B6D2F1 host, acute GVHD develops. Although it remains unclear what underlying mechanism is responsible for the distinct disease outcomes using these 2 injection protocols it is believed that the cytokines expressed by the cells contained within the DBA/2 splenocyte graft favor the development of chronic GVHD while the cytokines expressed by the cells contained within the C57BI/6 splenocyte graft favor the development of acute GVHD. Reagents which interfere with T cell interactions with antigen presenting cells (e.g. dendritic cells, macrophages, B cells: APC) effectively block both acute and chronic GVHD.
A number of lines of evidence suggest that acute GVHD is a Th1 mediated disease (Krenger and Ferrara, Immunol. Res. 15: 50-73 (1996), Williamson et al., J. Immunol, 157: 689-699 (1996)). Cytotoxic activity by CD4+ and CD8+ T cells, by natural killer (NK) cells, and by activated granulocytes such as macrophages, is a well defined consequence of Th1 mediated T cell response, and shows a characteristic dependence on the expression of II-2 and IFN-γ, which are typical Th1 cytokines. Such cytotoxicity is a defining characteristic of acute GVHD. Furthermore, reagents which block critical cytokines involved in Th1 T cell differentiation, such as mAbs to II-2 and II-12, block the development of acute GVHD. Cytotoxicity can be directly cellular (e.g. by phagocytosis of host cells) or by the action of secreted cytokines such as TNF which can induce apoptosis, or cell death. The consequence of donor anti-host cytotoxicity can be seen in a number of ways. First, host lymphocytes are rapidly destroyed, such that mice experiencing acute GVHD are profoundly immunosuppressed. Secondly, donor lymphocytes become engrafted and expand in the host spleen, and their cytotoxic activity can be directly measured in vitro by taking advantage of cell lines which express the host antigens that can be recognized (as foreign) by the donor cells. For example, cell lines expressing the appropriate antigens can be labeled with radioactive chromium51 isotope. Release of this radioactive isotope into the culture media is evidence of the death of the labeled cell. Third, the disease becomes lethal as additional tissues and cell populations are destroyed, and therefore survivorship is a measurable consequence of disease.
Chronic GVHD appears to be a Th2 T cell mediated disease (De Wit et al., J. Immunol. 150: 361-366 (1993)). In the mouse model the development of the disease is dependent on the Th2 cytokine 114, and can be blocked by treating with anti-114 mAb. Such treatment blocks the expansion of host B cells, and the concomitant hyper-Ig production. The development of GVHD can be followed in a number of ways. The expansion of the donor T cell and host B cell populations can be measured by the spleen index, which is the ratio of spleen weight to body weight, normalized to control (non-diseased) mice. The activation of B cells in diseased mice can be measured using analyses of B cell activation markers. Finally, the effects of B cell activation can be seen in the levels of Ig in circulation (eg. in serum) or produced by cultures of host splenocytes harvested several weeks after disease induction. Circulating Ig in diseased animals will contain anti-self antibodies. Ultimately, diseased animals succumb to kidney and other organ failure due to accumulated Ig deposition, and therefore survivorship is a relevant measure of disease activity.
We now show that a monoclonal antibody specific for TWEAK effectively and specifically blocks aspects of the development of GVHD, using the mouse model of chronic GVHD. The block in development of chronic GVHD is shown as a reduction in the spleen index, the loss of activation markers on host B cells, and reduced Ig production in the anti-TWEAK treated animals.